Vaccines containing ribavirin and methods of use thereof

ABSTRACT

The present invention relates to compositions and methods for enhancing the effect of vaccines in animals, such as domestic, sport, or pet species, and humans. More particularly, the use of Ribavirin as an adjuvant to a vaccine protocol and compositions having Ribavirin and an antigen are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/719,619 filed on Nov. 20, 2003, which is a continuation of U.S.patent application Ser. No. 10/104,966 filed on Mar. 22, 2002, now U.S.Pat. No. 6,680,059 which is a continuation of U.S. patent applicationSer. No. 09/705,547 filed on Nov. 3, 2000, now abandoned both of whichclaim the benefit of priority of U.S. provisional patent application No.60/229,175, filed Aug. 29, 2000. The aforementioned applications arehereby expressly incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for enhancingthe effect of vaccines in animals, such as domestic, sport, or petspecies, and humans. More particularly, preferred embodiments concernthe use of Ribavirin as an adjuvant and compositions having Ribavirinand an antigen.

BACKGROUND OF THE INVENTION

The use of vaccines to prevent disease in humans, farm livestock, sportsanimals, and household pets is a common practice. Frequently, however,the antigen used in a vaccine is not sufficiently immunogenic to raisethe antibody titre to levels that are sufficient to provide protectionagainst subsequent challenge or to maintain the potential for mountingthese levels over extended time periods. Further, many vaccines arealtogether deficient in inducing cell-mediated immunity, which is aprimary immune defense against bacterial and viral infection. Aconsiderable amount of research is currently focussed on the developmentof more potent vaccines and ways to enhance the immunogenicity ofantigen-containing preparations. (See e.g., U.S. Pat. Nos. 6,056,961;6,060,068; 6,063,380; and Li et al., Science 288:2219-2222 (2000)).

Notorious among such “weak” vaccines are hepatitis B vaccines. Forexample, recombinant vaccines against hepatitis B virus such asGenhevacb (Pasteur Merieux Serums et Vaccines, 58, Avenue Leclerc 69007Lyon, France), Engerixb (Smith, Kline and Symbol French), andRecombivaxhb (Merck, Sharp, and Dhome) are effective only after at leastthree injections at 0, 30, and 60 or 180 days, followed by an obligatorybooster after one year. (Chedid et al., U.S. Pat. No. 6,063,380).Additionally, many subjects receiving these vaccines respond poorly, ifat all. Because many regions of the world are endemic for HBV infection,the poorly immunogenic character of existing HBV vaccines has become anextremely serious problem.

To obtain a stronger, humoral and/or cellular response, it is common toadminister a vaccine in a material that enhances the immune response ofthe patient to the antigen present in the vaccine. The most commonlyused adjuvants for vaccine protocols are oil preparations and alum.(Chedid et al., U.S. Pat. No. 6,063,380). A greater repertoire of safeand effective adjuvants is needed.

Nucleoside analogs have been widely used in anti-viral therapies due totheir capacity to reduce viral replication. (Hosoya et al., J. Inf.Dis., 168:641-646 (1993)). Ribavirin(1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a syntheticguanosine analog that has been used to inhibit RNA and DNA virusreplication. (Huffman et al., Antimicrob. Agents. Chemother., 3:235(1973); Sidwell et al., Science, 177:705 (1972)). Ribavirin has beenshown to be a competitive inhibitor of inositol mono-phosphate (IMP)dehydrogenase (IMPDH), which converts IMP to IMX (which is thenconverted to GMP). De Clercq, Anti viral Agents: characteristic activityspectrum depending on the molecular target with which they interact,Academic press, Inc., New York N.Y., pp. 1-55 (1993). Intracellularpools of GTP become depleted as a result of long term Ribavirintreatment.

In addition to antiviral activity, investigators have observed that afew guanosine analogs have an effect on the immune system. (U.S. Pat.Nos. 6,063,772 and 4,950,647). Ribavirin has been shown to inhibitfunctional humoral immune responses (Peavy et al., J. Immunol.,126:861-864 (1981); Powers et al., Antimicrob. Agents. Chemother.,22:108-114 (1982)) and IgE-mediated modulation of mast cell secretion.(Marquardt et al., J. Pharmacol. Exp. Therapeutics, 240:145-149 (1987)).Some investigators report that a daily oral therapy of Ribavirin has animmune modulating effect on humans and mice. (Hultgren et al., J. Gen.Virol., 79:2381-2391 (1998) and Cramp et al., Gastron. Enterol.,118:346-355 (2000)). Nevertheless, the current understanding of theeffects of Ribavirin on the immune system is in its infancy.

SUMMARY OF THE INVENTION

It has been discovered that Ribavirin can be used as an adjuvant toenhance an immune response to an antigen. Embodiments described hereininclude “strong” vaccine preparations that comprise an antigen andRibavirin. Generally, these preparations have an amount of Ribavirinthat is sufficient to enhance an immune response to the antigen. Otheraspects of the invention include methods of enhancing the immuneresponse of an animal, including a human, to an antigen. By oneapproach, for example, an animal in need of a potent immune response toan antigen is identified and then is provided an amount of Ribavirintogether with the antigen that is effective to enhance an immuneresponse in the animal. In some methods, the Ribavirin and the antigenare provided in combination and in others, the Ribavirin and the antigenare provided separately. Thus, several embodiments concern themanufacture and use of vaccine preparations having Ribavirin and anantigen.

Preferred vaccine compositions comprise Ribavirin and a hepatitis viralantigen. The antigen can be a peptide or nucleic acid-based (e.g., a RNAencoding a peptide antigen or a construct that expresses a peptideantigen when introduced to a subject). HBV antigens that are suitableinclude, for example, hepatitis B surface antigen (HBsAg), hepatitiscore antigen (HBcAg), hepatitis e antigen (HBeAg), and nucleic acidsencoding these molecules. Compositions having Ribavirin and an antigenfrom the hepatitis A virus (HAV) or Ribavirin and a nucleic acidencoding an antigen from HAV are also embodiments. Still further,compositions having Ribavirin and an antigen from the hepatitis C virus(HCV) or Ribavirin and a nucleic acid encoding an antigen from HCV areembodiments.

Furthermore, compositions having a mixture of the antigens above areembodiments of the present invention. For example, some compositionscomprise a HBV antigen, a HAV antigen, and Ribavirin or a HBV antigen, aHCV antigen, and Ribavirin or a HAV antigen, a HCV antigen, andRibavirin or a HBV antigen, a HAV antigen, a HCV antigen, and Ribavirin.Other embodiments comprise Ribavirin and a nucleic acid encoding amixture of the antigens described above. Some embodiments also includeother adjuvants, binders, emulsifiers, carriers, and fillers, as knownin the art, including, but not limited to, alum, oil, and othercompounds that enhance an immune response.

Preferred methods involve providing an animal in need with a sufficientamount of Ribavirin and a hepatitis viral antigen (e.g., HBV antigen,HAV antigen, HCV antigen a nucleic acid encoding one of these antigensor any combination thereof). Accordingly, one embodiment includesidentifying an animal in need of an enhanced immune response to ahepatitis viral antigen (e.g., an animal at risk or already infectedwith a hepatitis infection) and providing to said animal an amount ofRibavirin that is effective to enhance an immune response to thehepatitis viral antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the humoral response to 10 and 100 μgrecombinant Hepatitis C virus (HCV) non structural 3 protein (NS3), asdetermined by mean end point titres, when a single dose of 1 mg ofRibavirin was co-administered.

FIG. 2 is a graph showing the humoral response to 20 μg recombinantHepatitis C virus (HCV) non structural 3 protein (NS3), as determined bymean end point titres, when a single dose of 0.1, 1.0, or 10 mg ofRibavirin was co-administered.

FIG. 3 is a graph showing the effects of a single dose of 1 mg Ribavirinon NS3-specific lymph node proliferative responses, as determined by invitro recall responses.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that compositions comprising Ribavirin and anantigen can boost an animal's immune response to the antigen. That is.Ribavirin can be used as an “adjuvant,” which for the purposes of thisdisclosure, refers to a compound that has the ability to enhance theimmune response to a particular antigen. Such adjuvant activity ismanifested by a significant increase in immune-mediated protectionagainst the antigen, and was demonstrated by an increase in the titer ofantibody raised to the antigen and an increase in proliferative T cellresponses.

Several vaccine preparations that comprise Ribavirin and an antigen aredescribed herein. Vaccine formulations containing Ribavirin can varyaccording to the amount of Ribavirin, the form of Ribavirin, and thetype of antigen. The antigen can be a peptide or a nucleic acid (e.g., aRNA encoding a peptide antigen or a construct that expresses a peptideantigen when introduced into a subject). Preferred vaccine formulationscomprise Ribavirin and a hepatitis viral antigen (e.g., HBV antigen, HAVantigen, HCV antigen, a nucleic acid encoding these molecules, or anycombination thereof).

Methods of enhancing the immune response of an animal, including humans,to an antigen are also described herein. One method, for example,involves identifying an animal in need of an enhanced immune response toan antigen and providing the animal the antigen and an amount ofRibavirin that is effective to enhance an immune response to theantigen. Preferred methods involve providing the animal in need withRibavirin and a hepatitis antigen (e.g., HBV antigen, HAV antigen, HCVantigen, a nucleic acid encoding these molecules, or any combinationthereof). The section below describes the manufacture of vaccines havingRibavirin and an antigen in greater detail.

Vaccines Containing Ribavirin

The vaccines comprise Ribavirin and an antigen and may contain otheringredients including, but not limited to, adjuvants, binding agents,excipients such as stabilizers (to promote long term storage),emulsifiers, thickening agents, salts, preservatives, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. These vaccinepreparations are suitable for treatment of animals either as apreventive measure to avoid a disease or condition or as a therapeuticto treat animals already afflicted with a disease or condition.

The vaccine compositions can be manufactured in accordance withconventional methods of galenic pharmacy to produce medicinal agents foradministration to animals, e.g., mammals including humans. Ribavirin canbe obtained from commercial suppliers (e.g., Sigma and ICN). Ribavirinand/or the antigen can be formulated into the vaccine with and withoutmodification. For example, the Ribavirin and/or antigen can be modifiedor derivatized to make a more stable molecule and/or a more potentadjuvant. By one approach, the stability of Ribavirin and/or an antigencan be enhanced by coupling the molecules to a support such as ahydrophilic polymer (e.g., polyethylene glycol).

Many more ribavirin derivatives can be generated using conventionaltechniques in rational drug design and combinatorial chemistry. Forexample, Molecular Simulations Inc. (MSI), as well as many othersuppliers, provide software that allows one of skill to build acombinatorial library of organic molecules. The C2.Analog Builderprogram, for example, can be integrated with MSI's suite of Cerius2molecular diversity software to develop a library of ribavirinderivatives that can be used with the embodiments described herein.

By one approach, the chemical structure of Ribavirin is recorded on acomputer readable media and is accessed by one or more modeling softwareapplication programs. The C2.Analog Builder program in conjunction withC2Diversity program allows the user to generate a very large virtuallibrary based on the diversity of R-groups for each substituentposition, for example. Compounds having the same structure as themodeled Ribavirin derivatives created in the virtual library are thenmade using conventional chemistry or can be obtained from a commercialsource.

The newly manufactured Ribavirin derivatives are then screened incharacterization assays, which determine the extent of adjuvant activityof the molecule and/or the extent of its ability to modulate of animmune response. Some characterization assays may involve virtual drugscreening software, such as C2.Ludi. C2.Ludi is a software program thatallows a user to explore databases of molecules (e.g., Ribavirinderivatives) for their ability to interact with the active site of aprotein of interest (e.g., RAC2 or another GTP binding protein). Basedupon predicted interactions discovered with the virtual drug screeningsoftware, the Ribavirin derivatives can be prioritized for furthercharacterization in conventional assays that determine adjuvant activityand/or the extent of a molecule to modulate an immune response.

Example 1 describes a characterization assay that was used to evaluatethe adjuvant activity of Ribavirin.

EXAMPLE 1

This characterization assay can be used with any Ribavirin derivative orcombinations of Ribavirin derivatives to determine the extent ofadjuvant activity of the particular vaccine formulation. Accordingly,groups of three to five Balb/c mice (BK Universal, Uppsala, Sweden) wereimmunized i.p or s.c. (e.g., at the base of the tail) with 10 μg or 100μg of recombinant hepatitis C virus non-structural 3 (NS3) protein. TherNS3 was dissolved in phosphate buffered saline (PBS) alone or PBScontaining 1 mg Ribavirin (obtained from ICN, Costa Mesa, Calif.). Micewere injected with a total volume of 100 μl per injection.

At two and four weeks following i.p. immunization, all mice were bled byretro-orbital sampling. Serum samples were collected and analyzed forthe presence of antibodies to rNS3. To determine the antibody titer, anenzyme immunoassay (EIA) was performed. (See e.g., Hultgren et al., JGen Virol. 79:2381-91 (1998) and Hultgren et al., Clin. Diagn. Lab.Immunol. 4:630-632 (1997), both of which are herein expresslyincorporated by reference in their entireties). The antibody levels wererecorded as the highest serum dilution giving an optical density at 405nm more than twice that of non-immunized mice.

Mice that received 10 μg or 100 μg rNS3 mixed with 1 mg Ribavirin in PBSdisplayed consistently higher levels of NS3 antibodies. The antibodytiter that was detected by EIA at two weeks post-immunization is shownin FIG. 1. The vaccine formulations having 1 mg of Ribavirin and either10 μg or 100 μg of rNS3 induced a significantly greater antibody titerthan the vaccine formulations composed of only rNS3. This data providesevidence that Ribavirin has an adjuvant effect on the humoral immuneresponse of an animal and thus, enhances the immune response to theantigen.

The example below describes experiments that were performed to determinethe amount of Ribavirin that was needed to elicit an adjuvant effect.

EXAMPLE 2

To determine the dose of Ribavirin that is required to provide anadjuvant effect, the following experiments were performed. Groups ofmice (three per group) were immunized with a 20 μg rNS3 alone or amixture of 20 μg rNS3 and 0.1 mg, 1 mg, or 10 mg Ribavirin. The levelsof antibody to the antigen were then determined by EIA. The meanendpoint titers at weeks 1 and 3 were plotted and are shown in FIG. 2.It was discovered that the adjuvant effect provided by Ribavirin haddifferent kinetics depending on the dose of Ribavirin provided. Forexample, low doses (<1 mg) of Ribavirin were found to enhance antibodylevels at week one but not at week three, whereas, higher doses (1-10mg) were found to enhance antibody levels at week three. These datafurther verify that Ribavirin can be administered as an adjuvant andestablish that the dose of Ribavirin can modulate the kinetics of theadjuvant effect.

The example below describes another characterization assay that wasperformed to evaluate the ability of Ribavirin to modulate a cellularimmune response.

EXAMPLE 3

This characterization assay can be used with any Ribavirin derivative orcombinations of Ribavirin derivatives to determine the extent that aparticular vaccine formulation modulates a cellular immune response. Todetermine CD4⁺ T cell responses to Ribavirin-containing vaccine, groupsof mice were immunized s.c. with either 100 μg rNS3 in PBS or 100 μgrNS3 and 1 mg Ribavirin in PBS. The mice were sacrificed ten dayspost-immunization and their lymph nodes were harvested and drained. Invitro recall assays were then performed. (See e.g., Hultgren et al., JGen Virol. 79:2381-91 (1998) and Hultgren et al., Clin. Diagn. Lab.Immunol. 4:630-632 (1997), both of which are herein expresslyincorporated by reference in their entireties). The amount of CD4⁺ Tcell proliferation was determined at 96 h of culture by theincorporation of [³H] thymidine.

As shown in FIG. 2, mice that were immunized with 100 μg rNS3 mixed with1 mg Ribavirin had a much greater T cell proliferative response thanmice that were immunized with 100 μg rNS3 in PBS. This data providesevidence that Ribavirin can enhance a cellular immune response (e.g., bypromoting the effective priming of T cells).

The example below describes the use of Ribavirin in conjunction with acommercial vaccine preparation.

EXAMPLE 4

The adjuvant effect of Ribavirin was also tested when mixed with twodoses of a commercially available vaccine containing HBsAg and alum.(Engerix, SKB). Approximately 0.2 μg or 2 μg of Engerix vaccine wasmixed with either PBS or 1 mg Ribavirin in PBS and the mixtures wereinjected intra peritoneally into groups of mice (three per group). Abooster containing the same mixture was given on week four and all micewere bled on week six. The serum samples were diluted from 1:60 to1:37500 and the dilutions were tested by EIA, as described above, exceptthat purified human HBsAg (kindly provided by Professor DL Peterson,Commonwealth University, VA) was used as the solid phase antigen. Asshown in TABLE 1, vaccine formulations having Ribavirin enhanced theresponse to 2 μg of an existing vaccine despite the fact that thevaccine already contained alum. That is, by adding Ribavirin to asuboptimal vaccine dose (i.e., one that does not induce detectableantibodies alone) antibodies became detectable, providing evidence thatthe addition of Ribavirin allows for the use of lower antigen amounts ina vaccine formulation without compromising the immune response.

TABLE 1 End point antibody titer to HBsAg in EIA 0.02 μg Engerix 0.2 μgEngerix No Ribavirin 1 mg Ribavirin No Ribavirin 1 mg Ribavirin Week #1#2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 6 <60 <60 <60 <60 <60 <60 <60 <60 <60300 60 <60

Any antigen that can be used to generate an immune response in an animalcan be combined with Ribavirin so as to prepare the vaccines describedherein. That is, antigens that can be incorporated into such a vaccinecomprise bacterial antigens, fungal antigens, plant antigens, moldantigens, viral antigens, cancer cell antigens, toxin antigens, chemicalantigens, and self-antigens. Although many of these antigens aremolecules that induce a significant immune response without an adjuvant,Ribavirin can be administered in conjunction with or combined with“strong” or “weak” antigens to enhance the immune response. In addition,the use of Ribavirin as an adjuvant may allow for the use of loweramounts of vaccine antigens while retaining immunogenicity.

Preferred embodiments comprise Ribavirin and a viral antigen. Preferredviral antigens are hepatitis viral antigens. Vaccines can comprise, forexample, Ribavirin and an HBV antigen, HAV antigen, HCV antigen or anycombination of these antigens. Preferred viral antigens includehepatitis B surface antigen (HBsAg), hepatitis core antigen (HBcAg), andhepatitis E antigen (HBeAg).

For example, HCV vaccine embodiments comprise Ribavirin and a HCVpeptide of at least 3 consecutive amino acids of SEQ. ID. No.: 1. Thatis, a vaccine embodiment can have Ribavirin and a HCV peptide with alength of at least 3-10 consecutive amino acids, 10-50 consecutive aminoacids, 50-100 consecutive amino acids, 100-200 consecutive amino acids,200-400 consecutive amino acids, 400-800 consecutive amino acids,800-1200 consecutive amino acids, 1200-1600 consecutive amino acids,1600-2000 consecutive amino acids, 2000-2500 consecutive amino acids,and 2500-3011 consecutive amino acids of SEQ ID. No. 1. Preferred HCVvaccines comprise Ribavirin and a peptide of at least 3 consecutiveamino acids of HCV core protein (SEQ. ID. No. 2), HCV E1 protein (SEQ.ID. No. 3), HCV E2 protein (SEQ. ID. No. 4), HCV NS2 (SEQ. ID. No. 5),HCV NS3 (SEQ. ID. No. 6), HCV NS4A (SEQ. ID. No. 7), HCV NS4B (SEQ. ID.No. 8), or HCV NS5A/B (SEQ. ID. No. 9). That is, preferred HCV vaccinescan comprise Ribavirin and a peptide with a length of at least 3-10consecutive amino acids, 10-50 consecutive amino acids, 50-100consecutive amino acids, 100-200 consecutive amino acids, 200-400consecutive amino acids, 400-800 consecutive amino acids, and 800-1040consecutive amino acids of any one of (SEQ. ID. Nos. 2-9).

Similarly, preferred HBV vaccine embodiments comprise Ribavirin and aHBV peptide of at least 3 consecutive amino acids of HBsAg (SEQ. ID.No.: 10) or HBcAg and HBeAg (SEQ. ID. No. 11). That is, a vaccineembodiment can have Ribavirin and a HBV peptide with a length of atleast 3-10 consecutive amino acids, 10-50 consecutive amino acids,50-100 consecutive amino acids, 100-150 consecutive amino acids, 150-200consecutive amino acids, and 200-226 consecutive amino acids of eitherSEQ. ID. No. 10 or SEQ. ID. No. 11. Further, preferred HAV embodimentscomprise Ribavirin and a HAV peptide with a length of at least 3-10consecutive amino acids, 10-50 consecutive amino acids, 50-100consecutive amino acids, 100-200 consecutive amino acids, 200-400consecutive amino acids, 400-800 consecutive amino acids, 800-1200consecutive amino acids, 1200-1600 consecutive amino acids, 1600-2000consecutive amino acids, and 2000-2227 consecutive amino acids of SEQID. No. 12.

In addition to peptide antigens, nucleic acid-based antigens can be usedin the vaccine compositions described herein. Various nucleic acid-basedvaccines are known and it is contemplated that these compositions andapproaches to immunotherapy can be augmented by introducing Ribavirin(See e.g., U.S. Pat. No. 5,589,466, herein expressly incorporated byreference in its entirety).

By one approach, for example, a gene encoding a polypeptide antigen ofinterest is cloned into an expression vector capable of expressing thepolypeptide when introduced into a subject. The expression construct isintroduced into the subject in a mixture of Ribavirin or in conjunctionwith Ribavirin (e.g., Ribavirin is administered shortly after theexpression construct at the same site). Alternatively, RNA encoding apolypeptide antigen of interest is provided to the subject in a mixturewith Ribavirin or in conjunction with Ribavirin. Where thepolynucleotide is to be DNA, promoters suitable for use in variousvertebrate systems are well known. For example, for use in murinesystems, suitable strong promoters include RSV LTR, MPSV LTR, SV40 IEP,and metallothionein promoter. In humans, on the other hand, promoterssuch as CMV IEP can be used. All forms of DNA, whether replicating ornon-replicating, which do not become integrated into the genome, andwhich are expressible, can be used.

Preferred nucleic acid-based antigens include a nucleotide sequence ofat least 9 consecutive nucleotides of HCV (SEQ. ID. No. 13), HBV (SEQ.ID. No.: 14), or HAV (SEQ. ID. No. 15). That is, a nucleic acid basedantigen can comprise at least 9-25 consecutive nucleotides, 25-50consecutive nucleotides, 50-100 consecutive nucleotides, 100-200consecutive nucleotides, 200-500 consecutive nucleotides, 500-1000consecutive nucleotides, 1000-2000 consecutive nucleotides, 2000-4000consecutive nucleotides, 4000-8000 consecutive nucleotides, and8000-9416 consecutive nucleotides of any one of SEQ. ID. Nos.: 13-15 oran RNA that corresponds to these sequences.

The example below describes one approach for using a nucleic acid-basedantigen in conjunction with Ribavirin.

EXAMPLE 5

The following describes an approach to immunize an animal with a vaccinecomprising a nucleic acid-based antigen and Ribavirin. Five to six weekold female and male Balb/C mice are anesthetized by intraperitonealinjection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on theanterior thigh, and the quadriceps muscle is directly visualized. Onegroup of mice are injected with approximately 20 μg of an expressionconstruct having the gp-120 gene, driven by a cytomegalovirus (CMV)promotor and second group of mice are injected with approximately 5 μgof capped in vitro transcribed RNA (e.g., SP6, T7, or T3 (Ambion))encoding gp-120. These two groups are controls. A third group of mice isinjected with approximately 20 μg of the expression vector having thegp-120 gene and the CMV promoter mixed with 1 mg of Ribavirin and afourth group of mice is injected with approximately 5 μg of capped invitro transcribed RNA mixed with 1 mg Rbavirin. The vaccines areinjected in 0.1 ml of solution (PBS) in a 1 cc syringe through a 27gauge needle over one minute, approximately 0.5 cm from the distalinsertion site of the muscle into the knee and about 0.2 cm deep. Asuture is placed over the injection site for future localization, andthe skin is then closed with stainless steel clips.

Blood samples are obtained prior to the injection (Day 0) and up to morethan 40 days post injection. The serum from each sample is seriallydiluted and assayed in a standard ELISA technique assay for thedetection of antibody, using recombinant gp-120 protein made in yeast asthe antigen. Both IgG and IgM antibodies specific for gp-120 will bedetected in all samples, however, groups three and four, which containedthe Ribavirin, will exhibit a greater immune response to the gp-120 asmeasured by the amount and/or titer of antibody detected in the sera.

Many other ingredients can be present in the vaccine. For example, theRibavirin and antigen can be employed in admixture with conventionalexcipients (e.g., pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral, enteral (e.g., oral) ortopical application that do not deleteriously react with the Ribavirinand/or antigen). Suitable pharmaceutically acceptable carriers include,but are not limited to, water, salt solutions, alcohols, gum arabic,vegetable oils, benzyl alcohols, polyetylene glycols, gelatine,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Many more suitablecarriers are described in Remmington's Pharmaceutical Sciences, 15thEdition, Easton:Mack Publishing Company, pages 1405-1412 and1461-1487(1975) and The National Formulary XIV, 14th Edition,Washington, American Pharmaceutical Association (1975), herein expresslyincorporated by reference in their entireties. Vaccines can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, coloring, flavoring and/oraromatic substances and the like that do not deleteriously react withRibavirin or the antigen.

The effective dose and method of administration of a particular vaccineformulation can vary based on the individual patient and the type andstage of the disease, as well as other factors known to those of skillin the art. Therapeutic efficacy and toxicity of the vaccines can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population). The data obtained from cell culture assays andanimal studies can be used to formulate a range of dosage for human use.The dosage of the vaccines lies preferably within a range of circulatingconcentrations that include the ED50 with no toxicity. The dosage varieswithin this range depending upon the type of Ribavirin derivative andantigen, the dosage form employed, the sensitivity of the patient, andthe route of administration.

Since Ribavirin has been on the market for several years, many dosageforms and routes of administration are known. All known dosage forms androutes of administration can be provided within the context of theembodiments described herein. Preferably, an amount of Ribavirin that iseffective to enhance an immune response to an antigen in an animal canbe considered to be an amount that is sufficient to achieve a bloodserum level of antigen approximately 0.25-12.5 μg/ml in the animal,preferably, about 2.5 μg/ml. In some embodiments, the amount ofRibavirin is determined according to the body weight of the animal to begiven the vaccine. Accordingly, the amount of Ribavirin in a vaccineformulation can be from about 0.1-6.0 mg/kg body weight. That is, someembodiments have an amount of Ribavirin that corresponds toapproximately 0.1-1.0 mg/kg, 1.1-2.0 mg/kg, 2.1-3.0 mg/kg, 3.1-4.0mg/kg, 4.1-5.0 mg/kg, 5.1, and 6.0 mg/kg body weight of an animal. Moreconventionally, the vaccines contain approximately 0.25 mg-2000 mg ofRibavirin. That is, some embodiments have approximately 250 μg, 500 μg,1 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g,1.9 g, and 2 g of Ribavirin.

Vaccines comprising various antigens and amounts of these antigens havebeen provided to animals for many years. Thus, conventional vaccinepreparations can be modified by adding an amount of Ribavirin that issufficient to enhance an immune response to the antigen. That is,existing conventional vaccine formulations can be modified by simplyadding Ribavirin to the preparation or by administering the conventionalvaccine in conjunction with Ribavirin (e.g., shortly before or afterproviding the antigen). As one of skill in the art will appreciate, theamount of antigens in a vaccine can vary depending on the type ofantigen and its immunogenicity. The amount of antigens in the vaccinescan vary accordingly. Nevertheless, as a general guide, the vaccines canhave approximately 0.25 mg-2000 mg of an antigen (e.g., a hepatitisviral antigen).

In some approaches described herein, the exact amount of Ribavirinand/or antigen is chosen by the individual physician in view of thepatient to be treated. Further, the amounts of Ribavirin can be added incombination to or separately from the same or equivalent amount ofantigen and these amounts can be adjusted during a particularvaccination protocol so as to provide sufficient levels in light ofpatient-specific or antigen-specific considerations. In this vein,patient-specific and antigen-specific factors that can be taken intoaccount include, but are not limited to, the severity of the diseasestate of the patient, age, and weight of the patient, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy.

Routes of administration of the vaccines described herein include, butare not limited to, transdermal, parenteral, gastrointestinal,transbronchial, and transalveolar. Transdermal administration can beaccomplished by application of a cream, rinse, gel, etc. capable ofallowing Ribavirin and antigen to penetrate the skin. Parenteral routesof administration include, but are not limited to, electrical or directinjection such as direct injection into a central venous line,intravenous, intramuscular, intraperitoneal, intradermal, orsubcutaneous injection. Gastrointestinal routes of administrationinclude, but are not limited to, ingestion and rectal. Transbronchialand transalveolar routes of administration include, but are not limitedto, inhalation, either via the mouth or intranasally.

Compositions having Ribavirin and an antigen that are suitable fortransdermal administration include, but are not limited to,pharmaceutically acceptable suspensions, oils, creams, and ointmentsapplied directly to the skin or incorporated into a protective carriersuch as a transdermal device (“transdermal patch”). Examples of suitablecreams, ointments, etc. can be found, for instance, in the Physician'sDesk Reference. Examples of suitable transdermal devices are described,for instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen,et al., herein expressly incorporated by reference in its entirety.

Compositions having Ribavirin and an antigen that are suitable forparenteral administration include, but are not limited to,pharmaceutically acceptable sterile isotonic solutions. Such solutionsinclude, but are not limited to, saline, phosphate buffered saline andoil preparations for injection into a central venous line, intravenous,intramuscular, intraperitoneal, intradermal, or subcutaneous injection.

Compositions having Ribavirin and an antigen that are suitable fortransbronchial and transalveolar administration include, but not limitedto, various types of aerosols for inhalation. Devices suitable fortransbronchial and transalveolar administration of these are alsoembodiments. Such devices include, but are not limited to, atomizers andvaporizers. Many forms of currently available atomizers and vaporizerscan be readily adapted to deliver vaccines having Ribavirin and anantigen.

Compositions having Ribavirin and an antigen that are suitable forgastrointestinal administration include, but not limited to,pharmaceutically acceptable powders, pills or liquids for ingestion andsuppositories for rectal administration.

Once the vaccine comprising Ribavirin and an antigen has been obtained,it can be administered to a subject in need to treat or preventdiseases. The next section describes methods that employ the vaccinesdescribed above.

Methods of Use of Vaccines that Contain Ribavirin

The vaccines containing Ribavirin and an antigen can be used to treatand prevent a vast spectrum of diseases and can enhance the immuneresponse of an animal to an antigen. As one of skill in the art willappreciate conventional vaccines have been administered to subjects inneed of treatment or prevention of bacterial diseases, viral diseases,fungal diseases, and cancer. Because the vaccines described hereininclude conventional vaccines, which have been modified by the additionof Ribavirin, the methods described herein include the treatment andprevention of a disease using a vaccine that comprises an antigen andRibavirin.

Preferred embodiments concern methods of treating or preventinghepatitis infection. In these embodiments, an animal in need is provideda hepatitis antigen (e.g., a peptide antigen or nucleic acid-basedantigen) and an amount of Ribavirin sufficient to exhibit an adjuvantactivity in said animal. Accordingly, an animal can be identified as onein need by using currently available diagnostic testing or clinicalevaluation. The range of hepatitis viral antigens that can be used withthese embodiments is diverse. Preferred hepatitis viral antigens includean HBV antigen, an HAV antigen, an HCV antigen, nucleic acids encodingthese antigens, or any combination thereof. Highly preferred embodimentsinclude an HBV antigen selected from the group consisting of hepatitis Bsurface antigen (HBsAg), hepatitis core antigen (HBcAg), and hepatitis Eantigen (HBeAg), in particular, the peptide and nucleic acid-basedantigens describes supra. The Ribavirin and antigen can be providedseparately or in combination, and other adjuvants (e.g., oil, alum, orother agents that enhance an immune response) can also be provided tothe animal in need. Thus, preferred embodiments include methods oftreating or preventing hepatitis in an animal (e.g., HBV) by identifyingan infected animal or an animal at risk of infection and providing saidanimal a hepatitis antigen (e.g., HBsAg, HBcAg, and HBeAg) and an amountof Ribavirin sufficient to exhibit adjuvant activity.

Other embodiments include methods of enhancing an immune response to anantigen by providing an animal in need with an amount of Ribavirin thatis effective to enhance said immune response. In these embodiments, ananimal in need of an enhanced immune response to an antigen isidentified by using currently available diagnostic testing or clinicalevaluation. Oftentimes these individuals will be suffering from adisease (e.g., bacterial, fungal, mold, viral, or cancer) or are at riskfrom contracting the disease. However, an animal in need of an enhancedimmune response can be an animal that has been poisoned (e.g., bit by apoisonous insect or animal) or that has been exposed to a toxin or othertoxic compound. Once identified, these animals are provided anappropriate antigen and an amount of Ribavirin effective to enhance animmune response in the animal.

As above, the hepatitis viral antigens that can be used with theseembodiments include, but are not limited to, an HBV antigen, an HAVantigen, an HCV antigen, a nucleic acid encoding these molecules, or anycombination thereof. Highly preferred embodiments include an HBV antigenselected from the group consisting of hepatitis B surface antigen(HBsAg), hepatitis core antigen (HBcAg), and hepatitis E antigen(HBeAg), in particular, the peptide and nucleic acid-based antigensdescribed supra. The Ribavirin and antigen can be provided separately orin combination, and other adjuvants (e.g., oil, alum, or other agentsthat enhance an immune response) can also be provided to the animal inneed. Thus, preferred embodiments include methods of enhancing an immuneresponse to a hepatitis antigen (e.g., HBV) by identifying an animal inneed and providing the animal a hepatitis antigen (e.g., HBsAg, HBcAg,and HBeAg) and an amount of Ribavirin that is effective to enhance animmune response in the animal.

Although the invention has been described with reference to embodimentsand examples, it should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims. All referencescited herein are hereby expressly incorporated by reference.

1. A method of producing a detectable amount of antibody specific for ahepatitis viral antigen in a subject from an amount of hepatitis viralantigen that does not produce detectable antibodies, consistingessentially of: mixing said hepatitis viral antigen with an effectiveamount of ribavirin to produce an immunogenic composition; andproviding, in a single administration, said immunogenic composition tosaid subject.
 2. The method of claim 1, wherein said viral antigen is ahepatitis B viral antigen.
 3. The method of claim 2, wherein saidhepatitis B viral antigen is a hepatitis B surface antigen.
 4. Themethod of claim 1, further comprising measuring the titer of antibodiesspecific for said hepatitis viral antigen.
 5. The method of claim 1,further comprising identifying a subject in need of antibodies specificfor a hepatitis viral antigen.
 6. The method of claim 2, furthercomprising identifying a subject in need of antibodies specific for ahepatitis viral antigen.
 7. The method of claim 1, wherein saidimmunogenic composition is provided to said subject intranasally.
 8. Themethod of claim 1, wherein said immunogenic composition is provided tosaid subject transdermally.
 9. The method of claim 1, wherein saidimmunogenic composition comprises an oil.
 10. The method of claim 1,wherein said immunogenic composition is formulated for use with aninjection device.
 11. The method of claim 10, wherein said injectiondevice is an electrical injection device.
 12. The method of claim 10,wherein said injection device is a needleless injection device.